Protein analysis is the foundation of modern biological research. Investigations of the expression and regulation of critical protein factors in biological processes and their applications in pharmaceutical and clinical studies provide vital information for experimental, pharmaceutical and clinical research of the pathogenesis of diseases and their prevention, diagnosis and treatments.
The recently patented Zestern technique (U.S. Pat. Nos. 8,293,487, 8,563,256 and 8,722,345) is an improvement of traditional methods of immunoblot-based protein analysis. While the protein samples are analyzed following a traditional immunoblotting process before detection, an additional step of elution is added in Zestern analysis to ensure the specificity of the assay. The antibody or antibody complex bound to the antigen of interest can be specifically competed out by competing molecule into elution solution. The amount of the eluted antibody or antibody complex in elution solution reflect the amount of antigen of interest in the sample to be analyzed. The total amount of eluted antibody or antibody complex can be quantified directly in solution, representing another advantage of Zestern analysis over traditional immunoblotting methods.
While Zestern analysis demonstrates advantage over traditional immunoblot methods for its simplicity and suitability for high throughput analysis, it poses new demand for suitable devices, as current existing devices for traditional immunoblot methods are not designed to meet the need of Zestern analysis, especially for high throughput purpose.
In traditional immunoblot analysis, represented by Western blot analysis, several types of membranes have been used and well optimized for immunoblot analysis. These membranes include both nitrocellulose membrane and PVDF membrane.
In traditional immunoblot analysis, the signal is detected on the very spot where the antibody or antibody complex bound to the antigen of interest on the membrane. This requires the membrane to be smooth and continuous to facilitate comparison of the detection results.
On the contrary, in Zestern analysis, antibody or antibody complex is liberated from the very spot where antibody or antibody complex bound to the antigen of interest by the competing molecule. The antibody or antibody complex is eluted from each spot respectively for quantification. Clearly, in Zestern analysis, the membrane cannot be continuous among protein samples. It must be separated from each other to allow elution of antibody or antibody complex for each protein sample separately, preventing cross-contamination of the signal from each other.
In Zestern analysis, for the membrane per se, there is no requirement regarding the shape or other physical characteristics of the membrane, as detection of the signal from each sample is not being processed on the membrane per se.
Multi-well plate has been widely used in biochemical assays and immunoblotting assays including ELISA assays. These multi-well plates include 6, 24, 96, and even 1536 well plate. It can also be referred as microtiter plate, microplate, or microwell plate.
Multi-well plate for ELISA assay generally has protein binding capacity at less than 1 μg/cm2. In contrast, a typical membrane for traditional immunoblotting, regardless of nitrocellulose or PVDF membrane, has protein binding capacity of 100 to 200 μg/cm2. While ELISA plate has achieved success in ELISA assay, its low protein binding capacity limits its application in Zestern analysis.
In the prototype of immunoblot analysis, Dot blot analysis, the antigen is applied directly to the membrane, and the membrane is allowed to dry in the air over an extended period of time. This practice may not to be the best form of sample application, for at least in routine practices, vacuum is applied to the membrane to increase the binding of the antigen to the membrane.
In Western blot analysis, antigen is trapped in the polyacrylamide gel during electrophoresis step. The trapped antigen is transferred to the membrane for blotting through a process called electroblotting.
In this invention, a multi-unit plate is designed both suitable for Zestern analysis and for electroblotting of the antigen to the membrane. Therefore, this invention provides solution to the unique demand of Zestern analysis for immunoblot analysis, especially for its application in multi-unit plate format. It also significantly increase the amount of antigen bound to the membrane due to electroblotting step.